Cancer causes a significant burden of disease around the world. In the United States, one of every three adults are expected to develop some form of cancer in their lifetime. Solid tumors are the most prevalent types of cancer. There is an unmet need in early diagnosis and prognosis of asymptomatic epithelial cancer patients. This need is particularly significant given that early diagnosis or prognosis results can significantly influence the course of disease by influencing treatment choices, thresholds and goals, and possibly enhance compliance.
Screening for epithelial cancers such as, for example, cancers of the upper aerodigestive track (oral cavity, larynx, pharynx, esophagus), stomach, lung, cervix, colon, penis, rectum or for pre-malignant lesions in the previously-mentioned sites, is a complex process that currently involves clinical, histologic and radiologic examination. Screening methods at the molecular level are needed to identify individuals that possess increased intrinsic risks for specific biologic pathways leading to premalignancies or cancer.
Equally important for the prevention or early diagnosis of epithelial cancers, risk assessment methods are needed to incorporate genomic findings to improve the prediction of a person's probability of developing the above-named cancers or premalignancies. At the population level, tobacco use is widely recognized as a major risk factor for epithelial cancer. Risk assessment methods are needed to screen large populations for increased cancer risk. Such genomic findings can have a significant impact on a person's decision to discontinue smoking. An additional area that genomic findings of genetic susceptibility can have an important impact in managing epithelial cancers is in the clinical development of novel chemotherapeutics. Targeted development according to one's unique genetic characteristics will lead to the development of the next generation of biologic therapies for cancer.
Tobacco use is a well-established risk factor or causative agent of epithelial cancers of the oral cavity, pharynx, larynx, esophagus, lung, stomach, cervix, and colon/rectum. According to the World Health Organization, tobacco use is associated with 5,000,000 deaths annually; due to a continuing trend of increased utilization globally, the number of deaths from tobacco-related diseases is expected to double in the next two decades. Therefore there is an unmet need for behavioral interventions and tobacco cessation activities that incorporate risk markers as deterrents to the initiation or continuation of tobacco products.
The human genome is continuously faced with the challenge of preserving its stability and integrity as cellular DNA is threatened by exogenous and endogenous sources. Environmental agents, such as ultraviolet light, ionizing radiation, toxic chemicals and carcinogens (e.g., those found in tobacco), and the like alter the structure of DNA leading to mutations that increase the risk of cancer. Cellular by-products of metabolism, like reactive oxygen species, are continual enemies of DNA integrity that create endogenous genetic damage. Genetic instability is further promoted by spontaneous changes in the DNA, such as deamination of cytosine which leads to the miscoding of uracil. Finally, despite the precision of the DNA machinery, errors occur in normal transcriptional processes that contribute to the overall instability.
The damage rendered from these agents results in various outcomes, most of which are adverse. Disturbances in DNA metabolism can result in cell-cycle arrest or apoptosis. Lesions may block the progress of replication, transcription, or chromosome segregation resulting in mutations or apoptosis (programmed cell death). The long term consequences of permanent mutations and chromosome aberrations include aging and cancer. Cancers and other diseases, of various types and severity, also result from inherited genetic defects.
In view of the various lesions encountered, one repair process is not sufficient to protect human DNA. As a result, evolution has created multiple, sophisticated DNA repair pathways that, collectively, protect the cell against most insults. The task of protection is divided into several primary repair pathways: direct reversal, base excision repair (BER), mismatch repair (MMR), homologous recombination and end joining, and nucleotide excision repair (NER). In the past decade, knowledge about these mechanisms has rapidly expanded regarding modality, function, and genetic etiology. To date, about 150 repair genes have been identified and described (Wood et al. (2005) Mutat. Res., 577: 275-283). However, the role of DNA repair in cancer development is not fully understood. Inherited defects in several DNA repair enzymes have shown to predispose individuals to cancer development, suggesting an important relationship between these mechanisms and cancer.
NER is the most versatile of the DNA repair pathways and is found in all the different kingdoms of life, including eubacteria, archaea, and eukaryotes (Batty and Wood (1999) Gene, 241:193-204). In human cells, NER is responsible for repairing a multitude of lesions that distort the helix, interfere with Watson and Crick base pairing, and obstruct DNA transcription (Costa et al. (2003) Biochimie, 85:1083-1099). For example, two of the major helical distorting lesions targeted by NER are cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts, both of which are induced by UV light. The human syndrome, xeroderma pigmentosum (XP), which results in severe photosensitivity and a high incidence of skin cancer, is known to be caused by NER defects. Studies of this syndrome, utilizing XP patient cells, have led to identification of the genes encoding the proteins involved in NER (Costa et al. (2003), supra). These proteins comprise seven complementation groups, identified as XPA-G.
The basic NER process involves three major steps: 1) damage recognition and assembly of the incision complex, 2) dual DNA incision and damage excision, and 3) DNA repair synthesis and ligation (Dip et al. (2004) DNA Repair, 3:1409-1423). The core components of NER have been identified via cloning and the core reaction has been reconstituted. The core factors assemble into two large multi-enzyme machines: one, which recognizes DNA damage and performs the incision, and the second, which constructs the repair patch (Aboussekhra et al. (1995) Cell, 80:859-868; Mu et al. (1995) J. Biol. Chem., 270:2415-2418; Araujo et al. (2000) Genes Dev., 14:349-359; Huan et al. (1994) Proc. Natl. Acad. Sci. U.S.A., 91:12213-12217).
In the first step, damage recognition, the XPC-hHR23B complex is thought to be responsible for the initial detection of DNA lesions. XPC is a 125 kDa protein product of the XPC gene that associates with hHR23B, a 58 kDa homolog of the Rad23 protein in yeast (Masutani et al. (1994) EMBO J., 13:1831-1843). Centrin 2, an 18 kDa centrosome component is also found within the complex (Araki et al. (2001) J. Biol. Chem., 276:18665-18672). The hHR23B subunit protects XPC from proteolytic degradation; thus, all cellular XPC protein is complexed with hHR23B (Dip et al. (2004), supra; van der Spek et al. (1996) Nucl. Acids Res. 24:2551-2559).
The model that XPC is the first arriving factor is still under debate as some contest that other factors, such as XPA, are responsible for the initial lesion recognition (Wakasugi and Sancar (1999) J. Biol. Chem., 274:18759-18768). Recent hypotheses suggest that XPC-hHR23B does indeed act as the initial sensor, but it is not the sole factor responsible for lesion recognition (Dip et al. (2004), supra). Instead, Dip et al. suggest that NER machinery recognizes lesions via a bipartite process that involves two separate steps: recognition and proof-reading. Id. XPC identifies distortions in the DNA via interactions with bases unable to form normal hydrogen bonds, binding to them with high affinity. Id.
Once the lesion has been identified by XPC-hHR23B, TFIIH is recruited to the site via XPC's interaction with the XPB and p62 subunits (Yokoi et al. (2000) J. Biol. Chem., 275:9870-9875). TFIIH is composed of a total of nine polypeptides: XPB, XPD, p62, p52, p44, p34, cdk7, cyclin H, and MATT (Drapkin and Reinberg (1994) Trends Biochem. Sci., 19:504-508). TFIIH is hypothesized to complete the second step of damage recognition: proofreading (Dip et al. (2004), supra). First, TFIIH is loaded onto the damaged strand where it begins to unwind the DNA by 20-25 base pairs, utilizing two DNA helicases with complementary functions: XPD unwinds the DNA in 5′→3′ direction, while XPB unwinds the DNA in the opposite direction (Weeda et al. (1990) Cell, 62:777-791; Weber et al. (1990) EMBO J., 9:437-1447; Schaeffer et al. (1994) EMBO J., 13:2388-2392; Roy et al. (1994) Cell, 79:1093-1101). The arrested function of one helicase and the continued translocation of the other results in distortion of the helix, which is thought to further the recruitment of other NER factors and serve as verification that damage does, indeed, exist (Dip et al. (2004), supra). Without recognition of damage, ATP hydrolysis by TFIIH will occur and the existing factors will disassociate (Costa et al. (2003), supra).
Once TFIIH is bound, the XPA-RPA complex can be incorporated into the incision complex. XPA is a 36 kDa, Zn2+-finger protein that shows a binding affinity for damaged DNA and associates with other core NER factors (Dip et al. (2004), supra). XPA's affinity for damaged DNA led to the concept that it may be responsible for DNA recognition; however, multiple studies have shown that its affinity is lower and less selective than that of XPC, leading to the current model as previously discussed (Lao et al. (1999) Biochemistry, 38:3974-3984). RPA (replication protein A), composed of three subunits (70, 30, and 14 kDa), also shows an affinity for damaged DNA and is needed (as is XPA) to help TFIIH open the double helix around the lesion (Evans et al. (1997) EMBO J., 16:6559-6573; Mu et al. (1997) J. Biol. Chem., 272:28971-28979). The 70 kDa subunit of RPA, which possesses three DNA binding domains, is about 30 nucleotides in length; this roughly matches the gapped DNA in NER and is thought to confer protection to the undamaged DNA strand as well as recruit replication factors (Dip et al. (2004), supra; Kolpashchikov et al. (2001) Nucl. Acids Res., 29:373-379). An additional function of XPA-RPA is the interaction with the two site-specific endonucleases, XPG and XPF-ERCC-1, to ensure that they incise at the correct location and the un-damaged strand remains uncut (de Laat et al. (1998) Genes Dev., 12:2598-2609; Matsunaga et al. (1996) J. Biol. Chem., 271:11047-11050; Bessho et al. (1997) J. Biol. Chem., 272:3833-3837). RPA has been found to have an additional role in DNA synthesis, following excision, as it remains associated to the DNA substrate, as compared to the other core factors which are released (Dip et al. (2004), supra). In summary, the XPA-RPA complex is thought to double-check that the pre-incision complex design is correct-in assembly and location-prior to activation of the two endonucleases and subsequent incision. Id.
The final step in the assembly of the incision complex is the recruitment of XPG and XPF-ERCC1. XPG is thought to be recruited first, as it associates with the center of DNA damage in XPA cells, while XPF does not (Volker et al. (2001) Mol. Cell, 8:213-224). Interestingly, XPG is thought to already be present in the pre-incision complex, prior to XPA, due to its stabilizing interaction with TFIIH (Araujo et al. (2001) Mol. Cell. Biol., 21:2281-2291). Studies utilizing cells with mutations in XPA support this hypothesis as XPG was still found to be at the damaged DNA sites (Volker et al. (2001), supra). However, in these XPA deficient cells, XPG was not able to make its 3′ incision, suggesting that XPA, along with RPA, is necessary for activating the endonuclease (de Laat et al. (1998) Nucl. Acids Res., 26:4146-4152). This suggests that the three factors, XPG, XPA, and RPA work together to bind to DNA (Reardon and Sancar (2003) Genes Dev., 17:2539-2551; Riedl et al. (2003) EMBO J., 22:5293-5303).
The XPG gene encodes a structure-specific 3′ endonuclease that 45 cleaves substrates containing bubbles, stem-loops, and splayed arms 46-50 as well as single strand overhangs from duplex DNA (Habraken et al. (1995) J. Biol. Chem., 270:30194-30198). Incisions are always made in one strand of duplex DNA, at the 3′ boundary of the open DNA complex. In NER, the XPG-encoded endonuclease has an additional function, an architectural one, as it is also required for the formation of the complete open complex (Evans et al. (1997), supra; Mu et al. (1997), supra).
The XPF-ERCC1 complex is the last factor incorporated into the incision complex (Volker et al. (2001), supra; Wakasugi and Sancar (1998) Proc. Natl. Acad. Sci. U.S.A., 95:6669-6674; Mu et al. (1996) J. Biol. Chem., 271:8285-8294). XPF-ERCC1 encodes a structure-specific 5′ endonuclease that cleaves similar lesions to the 3′ endonuclease (Bessho et al. (1997), supra; Sijbers et al. (1996) Cell, 86:811-822; de Laat et al. (1998) J. Biol. Chem., 273:7835-7842). Additionally, this endonuclease has been shown to participate in recombination repair; it is needed to cleave non-homologous 3′ DNA tails protruding from heteroduplex intermediates (Dip et al. (2004), supra; Adair et al. (2000) EMBO J., 19:3771-3778). The XPF subunit is responsible for the incising function as it contains a conserved nuclease motif, while the ERCC-1 subunit acts to stabilize XPF and interacts with XPA, linking the heterodimer to the NER complex (Matsunaga et al. (1996), supra; Wakasugi et al. (1997) J. Biol. Chem., 272: 6030-16034).
Once the incision complex is complete, incision and removal of the damaged DNA (the second step in NER), may occur. In vitro experiments have suggested that the catalytic activity of the endonucleases is inhibited by TFIIH, in the absence of ATP; the addition of ATP reverses this inhibition, allowing incision to occur (Costa et al. (2003), supra). The 3′ endonuclease incision occurs first, followed by the 5′ endonuclease. XPG activity can continue in the absence of XPF-ERCC1, but XPF-ERCC1, although its catalytic activity does not rely on prior XPG-mediated incision, does require the presence of the XPG protein in the incision complex (Mu et al. (1997), supra; Wakasugi et al. (1997), supra). The incisions occur asymmetrically around the lesion, with the 3′ incision three to nine nucleotides away from the lesion and the 5′ incision 15-25 nucleotides away from the lesion (Dip et al. (2004), supra).
The excised oligonucleotide, containing 24-32 nucleotides, is released, leaving a hydroxyl group at the 3′ end of the gap; this signifies the end of the second step. Without intending to be bound by scientific theory, at this point in time, most of the NER proteins have likely begun to disassemble and leave as the machinery for synthesis arrives. One core factor, RPA, remains at the site as it provides the template strand with protection from nucleases. The two DNA polymerases identified in the synthesis process are DNA Pol α and DNA Pol δ. PCNA and replication factor C (RFC), both proteins that act as processivity factors, are also required for DNA synthesis (Shivji et al. (1992) Cell, 69:367-374). In vitro synthesis utilizing these five factors (RPA, DNA Pol α or DNA pol δ, PCNA, and RFC) has been successful (Shivji et al. (1995) Biochemistry, 34:5011-5017). Finally, ligation of the 5′ end of the newly synthesized DNA to the original sequence occurs, it seems, via DNA ligase I.
It is important to note that cells possess a more efficient repair pathway termed transcription coupled repair (TCR). In the 1980's, it was observed that NER proceeds at a much quicker rate in actively transcribed mammalian genes than in transcriptionally silent genes (Friedberg (1996) Annu. Rev. Biochem., 65:15-42; Hanawalt (1994) Science, 266:1957-1958; Hanawalt and Spivak (1999) Advances in DNA Repair (eds. Dizdaroglu and Karakaya) Academic/Plenum Publishing, New York, pp. 169-179). The transcribed strand, specifically, is repaired at a much faster rate than the un-transcribed stand (Friedberg (1996), supra; Hanawalt (1994), supra; Hanawalt and Spivak (1999), supra). TCR is designated as one of two sub-pathways of NER; the other sub-pathway, global genome repair (GGR) was described in the previous paragraphs. Unlike GGR, XPC-hHR23B is not necessary in TCR (Batty and Wood (1999), supra). Instead, it is thought that the arrested RNA polymerase II recognizes damaged DNA as the initial sensor in TCR (Friedberg (2001) Nature, 1:22-33). TCR is essential for re-starting the RNA synthesis process, and in doing so, protects the cell from transcription blocking lesions that may result in apoptosis (Proietti et al. (2002) DNA Repair, 1:209-223).
Three syndromes are known to be caused by inherited defects in NER: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy (TTD). All three of these disorders are characterized by intense sun sensitivity (Bootsma et al. (2001) The Metabolic and Molecular Basis of Inherited Disease (eds. Scriver et al.), McGraw-Hill, New York, 1:677-703; Lehmann (2001) Genes Dev., 15:15-23). Persons with xeroderma pigmentosum experience a high incidence of UV light induced skin cancer, neurological problems, and internal tumors (Wood et al. (2001) Science, 291(5507):1284). This disorder may be the result of a mutation in any one of the seven XP genes: A-G. Cockayne Syndrome is the result of CSA or CSB gene mutations in the TCR pathway. This disorder is not associated with an increased risk for cancer and is characterized by impaired development (physical and neurological), which results in dwarfism and dysmyelination and premature aging. A combined xeroderma pigmentosum/Cockayne syndrome also exists and is thought to be the result of XPB, XBD, or XPG mutations (Lehmann (2001), supra; Friedberg et al. (1995) DNA Repair and Mutagenesis. (ASM Press, Washington; Bootsma et al. (1998) The Genetic Basis of Human Cancer (eds. Vogelstein and Kinzler) McGraw-Hill, New York pp. 245-274; Hoeijmakers (1994) Eur. J. Cancer, 30A:1912-1921; Rapin et al. (2000) Neurology, 55:1442-1449; Berneburg and Lehmann (2001) Adv. Genet., 43: 71-102). TTD is very similar to Cockayne Syndrome, but is accompanied by additional symptoms like scaly skin, and brittle hair and nails. Genetic analysis has revealed that XPD genes are defective in most cases, although XPB has also been shown to cause TTD (Weeda et al. (1997) Am. J. Hum. Genet., 60:320-329).
To date, the mechanisms of NER have been derived from studies that evaluate the pathway as it occurs on DNA substrates. Although this has been an incredible tool, enabling the core factors and reaction to be reconstituted, it does not represent the DNA as it exists in living cell and thus, our understanding of how NER functions in chromatin is limited (Reed (2005) DNA Repair, 4:909-918). Recent studies have attempted to gain insight about this aspect of NER, but they have provided only glimpses of information, setting the stage for future research.